Nucleic acid-free thermostable enzymes and methods of production thereof

ABSTRACT

The present invention provides thermostable enzymes, such as DNA polymerases and restriction endonucleases, that are substantially free from contamination with nucleic acids. The invention also provides methods for the production of these enzymes, and kits comprising these enzymes which may be used in amplifying or sequencing nucleic acid molecules, including through use of the polymerase chain reaction (PCR).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.09/866,816, filed May 30, 2001, now U.S. Pat. No. 6,531,301, which is acontinuation of U.S. application Ser. No. 09/229,967, filed Jan. 14,1999, now U.S. Pat. No. 6,245,533, which is a continuation of U.S.application No. 08/778,082, filed Jan. 2, 1997, now U.S. Pat. No.5,861,295.

FIELD OF THE INVENTION

The present invention is in the fields of molecular biology and proteinchemistry. Specifically, the invention provides compositions comprisingthermostable enzymes, particularly thermostable DNA polymerases andrestriction endonucleases, that are substantially free fromcontamination by nucleic acids, and methods for the production of suchthermostable enzymes. Compositions comprising the thermostable enzymesof the present invention may be used in a variety of applications,including amplification and sequencing of nucleic acids.

BACKGROUND OF THE INVENTION

A variety of techniques have been traditionally employed to facilitatethe preparation of intracellular proteins from microorganisms.Typically, the initial steps in these techniques involve lysis, ruptureor permeabilization of the bacterial cells, to disrupt the bacterialcell wall and allow release of the intracellular proteins into theextracellular milieu. Following this release, the desired proteins arepurified from the extracts, typically by a series of chromatographicsteps.

Several approaches have proven useful in accomplishing the release ofintracellular proteins from bacterial cells. Included among these arethe use of chemical lysis or permeabilization, physical methods ofdisruption, or a combination of chemical and physical approaches(reviewed in Felix, H., Anal. Biochem. 120:211-234 (1982)).

Chemical methods of disruption of the-bacterial cell wall that haveproven useful include treatment of cells with organic solvents such astoluene (Putnam, S. L., and Koch, A. L., Anal. Biochem. 63:350-360(1975); Laurent, S. J., and Vannier, F. S., Biochimie 59:747-750 (1977);Felix, H., Anal. Biochem. 120:211-234 (1982)), with chaeotropes such asguanidine salts (Hettwer, D., and Wang, H., Biotechnol. Bioeng.33:886-895 (1989)), with antibiotics such as polymyxin B (Schupp, J. M.,et al., BioTechniques 19:18-20 (1995); Felix, H., Anal. Biochem.120:211-234 (1982)), or with enzymes such as lysozyme or lysostaphin(McHenry, C. S, and Kornberg, A, J. Biol. Chem. 252(18):6478-6484(1977); Cull, M, and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990);Hughes, A. J., Jr., et al, J. Cell. Biochem. Suppl. 0 16(Part B):84(1992); Sambrook, J., et al., in: Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, p. 17.38 (1989); Ausubel, F. M., et al., in: Current Protocols inMolecular Biology, New York: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)).The permeabilization effects of these various chemical agents may beenhanced by concurrently treating the bacterial cells with detergentssuch as TRITON X-100®, sodium dodecylsulfate (SDS) or Brij 35 (Laurent,S. J., and Vannier, F. S., Biochimie 59:747-750 (1977); Felix, H., Anal.Biochem. 120:211-234 (1982); Hettwer, D., and Wang, H., Biotechnol.Bioeng. 33:886-895 (1989); Cull, M., and McHenry, C. S., Meth. Enzymol.182:147-153 (1990); Schupp, J. M., et al., BioTechniques 19:18-20(1995)), or with proteins or protamines such as bovine serum albumin orspermidine (McHenry, C. H., and Kornberg, A, J. Biol. Chem.252(18):6478-6484 (1977); Felix, H., Anal. Biochem. 120:211-234 (1982);Hughes, A. J., Jr., et al., J. Cell. Biochem. Suppl. 0 16(Part B):84(1992)).

In addition to these various chemical treatments, a number of physicalmethods of disruption have been used. These physical methods includeosmotic shock, e.g., suspension of the cells in a hypotonic solution inthe presence or absence of emulsifiers (Roberts, J. D., and Lieberman,M. W., Biochemistry 18:4499-4505 (1979); Felix, H., Anal. Biochem.120:211-234 (1982)), drying (Mowshowitz, D. B., Anal. Biochem. 70:94-99(1976)), bead agitation such as ball milling (Felix, H., Anal. Biochem.120:211-234 (1982); Cull, M., and McHenry, C. S., Meth. Enzymol.182:147-153 (1990)), temperature shock, e.g., freeze-thaw cycling(Lazzarini, R. A., and Johnson, L. D., Nature New Biol. 243:17-20(1975); Felix, H., Anal. Biochem. 120:211-234 (1982)), sonication (Amos,H., et al., J. Bacteriol. 94:232-240 (1967); Ausubel, F. M., et al., in:Current Protocols in Molecular Biology, New York: John Wiley & Sons, pp.4.4.1-4.4.7 (1993)) and pressure disruption, e.g., use of a frenchpressure cell (Ausubel, F. M., et al., in: Current Protocols inMolecular Biology, New York: John Wiley & Sons, pp. 16.8.6-16.8.8(1993)). Other approaches combine these chemical and physical methods ofdisruption, such as lysozyme treatment followed by sonication orpressure treatment, to maximize cell disruption and protein release(Ausubel, F. M., et al., in: Current Protocols in Molecular Biology, NewYork: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)).

These disruption approaches have several advantages, including theirability to rapidly and completely (in the case of physical methods)disrupt the bacterial cell such that the release of intracellularproteins is maximized. In fact, these approaches have been used in theinitial steps of processes for the purification of a variety ofbacterial cytosolic enzymes, including natural and recombinant proteinsfrom mesophilic organisms such as Escherichia coli, Bacillus subtilisand Staphylococcus aureus (Laurent, S. J., and Vannier, F. S., Biochimie59:747-750 (1977); Cull, M., and McHenry, C. S., Meth. Enzymol.182:147-153 (1990); Hughes, A. J., Jr., et al., J. Cell. Biochem. Suppl.0 16(Part B):84 (1992); Ausubel, F. M., et al., in: Current Protocols inMolecular Biology, New York: John Wiley & Sons, pp. 4.4.1-4.4.7 (1993)),as well as phosphatases, restriction enzymes, DNA or RNA polymerases andother proteins from thermophilic bacteria and archaea such as Thermusaquaticus, Thermus thermophilus, Thermus flavis, Thermus caldophilus,Thermotoga maritima, and Sulfolobus acidocaldarius (Shinomiya, T., etal., J. Biochem. 92(6):1823-1832 (1982); Elie, C., et al., Biochim.Biophys. Acta 951(2-3):261-267 (1988); Palm, P., et al., Nucl. AcidsRes. 21(21):4904-4908 (1993); Park, J. H., et al., Eur. J. Biochem.214(1):135-140 (1993); Harrell, R. A., and Hand, R. P., PCR Meth. Appl.3(6):372-375 (1994); Meyer, W., et al., Arch. Biochem. Biophys.319(1):149-156 (1995)).

However, these methods possess distinct disadvantages as well. Forexample, the physical methods by definition involve shearing andfracturing of the bacterial cell walls and plasma membranes. Theseprocesses thus result in extracts containing large amounts ofparticulate matter, such as membrane or cell wall fragments, which mustbe removed from the extracts, typically by centrifugation, prior topurification of the enzymes. This need for centrifugation limits thebatch size capable of being processed in a single preparation to that ofavailable centrifuge space; thus, large production-scale preparationsare impracticable if not impossible. Furthermore, physical methods, andmany chemical permeation techniques, typically result in the releasefrom the cells not only of the desired intracellular proteins, but alsoof undesired nucleic acids and membrane lipids (the latter particularlyresulting when organic solvents are used to permeabilize the cells).These undesirable cellular components also complicate the subsequentprocesses for purification of the desired proteins, as they increase theviscosity of the extracts (Sambrook, J., et al., in: Molecular Cloning:A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press, p. 17.38 (1989); Cull, M., and McHenry, C. S.,Meth. Enzymol. 182:147-153 (1990)), and bind with high avidity andaffinity to nucleic acid-binding proteins such as DNA polymerases, RNApolymerases and restriction enzymes.

These limitations have been partially overcome in the case of proteinsprepared from mesophilic bacteria. For example, mild chemical disruptionof E. coli, B. subtilis and Salmonella typhimurium has been conducted topermeabilize these cells, allowing free mobility of proteins across themembrane of the cells or resultant spheroplasts, but inducing retentionof most of the nucleic acids within the cell or spheroplast (Laurent, S.J., and Vannier, F. S., Biochimie 59:747-750 (1977); Hettwer, D., andWang, H., Biotechnol. Bioeng. 33:886-895 (1989); Cull, M., and McHenry,C. S., Meth. Enzymol. 182:147-153 (1990); Schupp, J. M., et al.,BioTechniques 19:18-20 (1995)). Similar approaches have also been takento limit the contamination of protein preparations from thermophilicbacteria, based on the demonstration that protein-permeable spheroplastcan be prepared from thermophiles like Thermus thermophilus by treatmentwith lysozyme with or without a detergent (Oshima, T., and Imahori, K.,Int. J. Syst. Bacteriol. 24(1):102-112 (1974)).

These approaches, however, are insufficient for preventing thecontamination of preparations of thermostable enzymes with DNA. Forexample, it has been reported by at least two different groups thatcommercially available preparations of Taq DNA polymerase arecontaminated with bacterial DNA (Rand, K. H., and Houck, H., Mol. CellProbes 4(6):445-450 (1990); Hughes, M. S., et al., J. Clin. Microbiol.32(8):2007-2008 (1994)), despite the use of gentle lysis procedures toliberate the enzyme from the cells. Furthermore, this contaminating DNAmay come not only from the organisms which are the source of the enzyme(Thermus aquaticus for natural Taq polymerase; E. coli for recombinantenzyme), but also from unknown organisms present in the reagents andmaterials used to purify the enzyme after its release from the cells(Rand, K. H., and Houck, H., Mol. Cell Probes 4(6):445-450 (1990);Hughes, M. S., et al., J. Clin. Microbiol. 32(8):2007-2008 (1994)).Since thermophilic enzymes such as DNA polymerases and restrictionenzymes are routinely used in automated techniques of DNA amplificationand sequencing, e.g., the Polymerase Chain Reaction (PCR), the presenceof contaminating DNA in the enzyme preparations is a significant problemsince it can give rise to spurious amplification or sequencing results.Thus, a need exists for preparations of thermostable enzymes that aresubstantially free of contamination by nucleic acids.

Various solutions to this problem have been suggested. For example, manyinvestigators routinely run “no-template controls” to allow subtractionof any spurious results from their experimental samples, or to determinethe extent of contamination of their enzyme preparations ((Rand, K. H.,and Houck, H., Mol. Cell Probes 4(6):445-450 (1990)). This approach,however, increases the resource and time requirements, and thus theexpense, of the assays, particularly in high-throughput settings such asin clinical applications of PCR. Also suggested have been methods ofeliminating nucleic acids in the enzyme preparations, such as treatmentof the preparations with DNAse or RNAse, restriction enzyme digestion,organic phase partitioning, or cesium chloride density gradientcentrifugation (Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153(1990); Rand, K. H., and Houck, H., Mol. Cell Probes 4(6):445-450(1990)), although these approaches have apparently not proved routinelysuccessful at removing contaminating DNA (Rand, K. H., and Houck, H.,Mol. Cell Probes 4(6):445-450 (1990)). Other methods of inactivating DNAhave been described, such as a method of heating samples at 60-100° C.in the presence of an acid at pH 3-4 (U.S. Pat. No. 5,417,862). However,while thermophilic enzymes are fairly resistant to these increasedtemperatures, they quickly lose enzymatic activity when exposed to a pHbelow about 5, thus precluding use of this method in purgingthermophilic enzyme preparations of nucleic acid contamination.

Thus, instead of attempting to remove nucleic acids from preparations ofthermostable enzymes, a more reasonable and successful approach would beto prevent contamination of the enzymes by nucleic acids from the outsetin the purification process. Such an approach would be two-pronged: 1)preventing release of nucleic acids from the bacterial cells duringpermeabilization of the cells to release the enzymes; and 2) preventingcontamination of the enzymes during the purification process itself.Furthermore, an optimal method would obviate the need for centrifugationin the process, thus allowing large-scale, and even continuous,production of nucleic acid-free thermophilic enzymes. The presentinvention provides such methods, and thermophilic enzymes produced bythese methods.

SUMMARY OF THE INVENTION

The present invention provides methods of making a thermostable enzymewhich is substantially free of nucleic acids, comprising permeabilizingbacterial cells to form spheroplasts and isolating the thermostableenzyme under conditions favoring the partitioning of nucleic acids fromthe thermostable enzyme. The invention is particularly directed tomethods wherein the permeabilization of the bacterial cells isaccomplished by contacting the cells with an aqueous solutioncomprising: a chaeotropic agent, preferably a guanidine salt and mostpreferably guanidine hydrochloride; and a nonionic detergent, preferablyTRITON X-100 or NP40. The invention is further directed to such methodswherein the conditions favoring the partitioning of nucleic acids fromthe thermostable enzyme comprise formation of an ultrafiltrate bymicrofiltration of the spheroplasts through a semi-permeable membrane,which is preferably a hydrophilic dialysis membrane, in the presence ofa salt, preferably ammonium sulfate or potassium chloride, andpurification of the thermostable enzyme from the ultrafiltrate,preferably by chromatography using sterile materials. The invention isparticularly directed to such methods wherein the bacterial cellsproviding the thermostable enzyme is a species of the genus Thermus,most preferably Thermus aquaticus, or a species of the genus Thermotoga,most preferably Thermotoga neapolitana or Thermotoga maritima. Theinvention is also directed to methods wherein the thermostable enzymebeing prepared is a thermostable DNA polymerase, most preferably Taq DNApolymerase, Tne DNA polymerase, Tma DNA polymerase, or a mutant,derivative or fragment thereof, or a thermostable restrictionendonuclease. The invention also provides the above-described enzymes,or mutants, derivatives or fragments thereof that are made according tothe methods provided. The invention is also directed to methods foramplifying or sequencing a nucleic acid molecule comprising contactingsaid nucleic acid molecule with a thermostable DNA polymerase madeaccording to the methods provided by the present invention. Theinvention also provides kits for amplifying or sequencing a nucleic acidmolecule comprising a carrier means having in close confinement thereinone or more container means, wherein a first such container meanscontains a thermostable enzyme, preferably a DNA polymerase or arestriction endonuclease, and most preferably Taq DNA polymerase, TneDNA polymerase, Tma DNA polymerase, or a mutant, derivative or fragmentthereof, made according to the methods of the present invention.

Other features and advantages of the present invention will be apparentto those skilled in the art from the following description of thepreferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the incorporation of ³H-thymidine by two Taq DNApolymerase preparations in the absence of exogenously added template,demonstrating that a preparation of Taq DNA polymerase made according tothe methods of the present invention (“New Taq”) is substantially freefrom contamination with DNA, while a preparation of Taq DNA polymerasemade according to standard methods (“Old Taq”) contains significantamounts of DNA.

FIG. 2 is a graph of the incorporation of ³H-thymidine by variouspreparations of Taq DNA polymerase in the absence of exogenously addedtemplate, demonstrating that a preparation of Taq DNA polymerase madeaccording to the methods of the present invention (“New Taq”) issubstantially free from contamination with DNA, while variouscommercially available preparations of Taq DNA polymerase containsignificant amounts of DNA.

DETAILED DESCRIPTION OF THE INVENTION

Overview

The present invention provides thermostable enzymes, such as DNApolymerases and restriction endonucleases, that are substantially freefrom contamination with nucleic acids. As used herein, the term“substantially free of nucleic acids” means an enzyme composition thatcomprises no nucleic acids, or that comprises nucleic acids below thelevel of detection, when assayed by standard biochemical assays fornucleic acids, which may include gel electrophoresis (e.g., agarose gelelectrophoresis coupled with ethidium bromide, acridine orange orHoechst staining), spectrophotometry (e.g., ultraviolet, atomicabsorption, NMR or mass spectrometry), chromatography (liquid, gas, HPLCor FPLC), or by functional assays for nucleic acids such as a measure ofincorporation of ³H-labeled nucleotides by the enzyme preparation in a“no-template” nucleic acid amplification control. These biochemical andfunctional assays are described in more detail below, and in Example 3.The invention also provides methods for the production of these enzymes,and compositions and kits comprising these enzymes which may be used inamplifying or sequencing nucleic acid molecules, including through useof the polymerase chain reaction (PCR).

Briefly summarized, the present invention utilizes a scheme comprisingpermeabilization of bacterial cells to form spheroplasts,microfiltration of the spheroplasts to form a microfiltrate,ultrafiltration of the microfiltrate to form an ultrafiltrate, andpurification of the enzyme from the ultrafiltrate, preferably byconventional liquid chromatography. The present invention 1) provides amethod of large-scale (>20 million unit) production of thermostableenzymes, including Taq DNA polymerase, which are substantially free ofDNA contamination; and 2) provides a scalable method for the productionof any desirable quantity of a thermostable enzyme.

The present methods are based in particular upon an optimized method ofchemical permeabilization of bacterial cells, which strips the bacterialcell wall and yields spheroplasts, and an optimized method ofmicrofiltration of the spheroplasts under conditions favoring therelease of thermostable enzymes, but inhibiting the release of nucleicacids, from the spheroplasts. The permeabilization process has beenoptimized to allow intracellular enzymes, including DNA polymerase andrestriction enzymes, to permeate the spheroplast membrane whilepreventing the cellular DNA from entering the permeation buffer. Thisapproach provides an initial extract that is enriched in enzyme and thatis relatively free of nucleic acids. The extract is then subjected tomicrofiltration under conditions (including precise definition of thevariables of salt, pH, and choice of membrane chemistry) favoringrelease of the enzyme from the spheroplasts while preventing cells, celldebris and/or nucleic acids from crossing the filtration membranebarriers. Following microfiltration and ultrafiltration, thermostableenzymes may be purified by standard techniques such as chromatography orelectrophoresis, to provide enzyme preparations which are substantiallyfree from contamination by nucleic acids.

Bacterial Cell Sources of Thermostable Enzymes

Thermostable enzymes (e.g., DNA polymerases or restriction enzymes) maybe prepared according to the methods of the present invention from avariety of thermophilic bacteria that are available commercially (forexample, from American Type Culture Collection, Rockville, Md.).Suitable for use as sources of thermostable enzymes are the thermophilicbacteria Thermus aquaticus, Thermus thermophilus, Thermococcuslitoralis, Pyrococcus furiosus, Pyrococcus woosii and other species ofthe Pyrococcus genus, Bacillus stearothermophilus, Sulfolobusacidocaldarius, Thermoplasma acidophilum, Thermus flavus, Thermus ruber,Thermus brockianus, Thermotoga neapolitana, Thermotoga maritima andother species of the Thermotoga genus, and Methanobacteriumthermoautotrophicum, and mutants of each of these species. It will beunderstood by one of ordinary skill in the art, however, that anythermophilic microorganism may be used as a source for preparation ofthermostable enzymes according to the methods of the present invention.Bacterial cells may be grown according to standard microbiologicaltechniques, using culture media and incubation conditions suitable forgrowing active cultures of the particular species that are well-known toone of ordinary skill in the art (see, e.g., Brock, T. D., and Freeze,H., J. Bacteriol. 98(1):289-297 (1969); Oshima, T., and Imahori, K, Int.J. Syst. Bacteriol. 24(1):102-112 (1974)).

Permeabilization of Bacterial Cells

In the initial steps of the present methods, the bacterial cells whichserve as the source for the thermostable enzymes are treated so as topermeabilize the cells by stripping away the cell walls and convertingthe cells into spheroplasts. Although a variety of techniques may beused for this permeabilization, such as treatment with enzymes (e.g.,lysozyme), the production of substantially nucleic acid-free enzymes bythe present invention requires the use of a permeabilization methodwhich will produce spheroplasts that retain DNA within the spheroplastwhile allowing free permeation of intracellular proteins (includingenzymes) across the spheroplast membrane. All procedures frompermeabilization to final purification of the enzymes should be carriedout at temperatures below normal room temperature, preferably at about4-15° C., most preferably at about 4-10° C., to prevent enzymedenaturation and loss of activity. Furthermore, all materials usedthroughout the present methods (i.e., reagents, salts, chromatographyresins, equipment) should be sterilized by heat or barrier sterilizationtechniques (as appropriate to the material to be sterilized), to preventthe contamination of the thermostable enzymes with nucleic acids.

This permeabilization is preferably accomplished by suspension of thebacterial cells in an aqueous solution comprising at least onechaeotropic agent and at least one nonionic detergent. Chaeotropicagents preferable for use in the methods of the present inventioninclude salts of guanidine or urea, most preferably guanidinehydrochloride. Any nonionic detergent may be used; most preferable areoctylphenoxypolyethoxyethanol nonionic surfactant (TRITON X-100®), Brij35, Tween 20 and Nonidet P40 (NP-40®), although other nonionicsurfactants and mixtures thereof, such as N-alkylglucosides,N-alkylmaltosides, glucamides, digitonin, deoxycholate,3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) orcetyltrimethylammoniumbromide (CTAB) may also be used in the presentcompositions. Reagents such as chaeotropes, detergents, buffer salts,etc., are available commercially, for example from Sigma Chemical Co.(St. Louis, Mo.).

For permeabilization, bacterial cells are suspended in a buffered saltsolution containing the chaeotrope(s) and detergent(s). Preferably, thesolution is an aqueous solution with a distilled, deionized water (dH₂O)base consisting of trishydroxymethylaminomethane (TRIS® base) at aconcentration of about 25-500 mM, preferably about 50-250 mM, morepreferably about 50-150 mM, and most preferably about 100 mM, at a pH ofabout 7.0-9.0, preferably about 7.5-9.0, more preferably about 8.0-8.5,and most preferably about 8.5 (pH at about 20-25° C.). The concentrationof the chaeotrope (e.g., guanidine hydrochloride) in the solution ispreferably about 300-1000 mM more preferably about 500-750 mM, and mostpreferably about 600 mM, while the nonionic detergent concentration ispreferably about 1-5% (vol/vol), more preferably about 1-2.5%, and mostpreferably about 1.8%. The permeabilization buffer solution may alsocomprise other components, such as protease inhibitors (e.g.,phenylmethylsulfonylfluoride, added at a final concentration of about0.5 mM), reducing agents (e.g., β-mercaptoethanol or most preferablydithiothreitol at a final concentration of about 1 mM), and chelatingagents (e.g., disodium ethylenediaminetetraacetic acid (Na₂EDTA), mostpreferably at a concentration of about 1.5 mM); this buffer compositionis referred to hereinafter as “permeabilization buffer.” It will beunderstood by one of ordinary skill in the art, however, that othersuitable buffer compositions may be substituted with equivalent effectin the permeabilization process, provided that the above-describedconcentrations of chaeotropes and detergents are used.

For permeabilization, bacterial cells are suspended in permeabilizationbuffer at a concentration of about 100-1000 g (wet weight) of cells perliter of solution, preferably about 250-1000 g/L, and most preferablyabout 500 g/L (cell density of about 1-5×10¹⁰ cells/gram, preferablyabout 2-5×10¹⁰ cells/gram, and most preferably about 2.5×10¹⁰cells/gram). The cell suspension is gently stirred, preferably viamagnetic or impeller stirring, in such a way as to prevent shearing andrupture of the cells. After about 30-60 minutes, most preferably about45 minutes, a protein-extracting salt is added to the suspension toenhance the permeation of the intracellular enzymes across thespheroplast membranes. Although any salt may be used in the presentinvention (except salts of toxic metals such as cadmium or other heavymetals), preferred salts include sodium chloride, potassium acetate,sodium acetate, ammonium acetate, ammonium chloride, ammonium sulfate orpotassium chloride, most preferably ammonium sulfate or potassiumchloride. Salt is added to the suspension at a concentration of about100-500 mM preferably about 100-300 mM, and most preferably about 400 mMfor salts with monoatomic cations (e.g., sodium chloride, potassiumacetate, sodium acetate, ammonium acetate, ammonium chloride orpotassium chloride) or about 200 mM for salts with diatomic cations(e.g., ammonium sulfate). Salt should be gradually added to thesolution, to provide for optimal solubilization. Following addition ofthe salt, the solution is mixed for about an additional 30-60 minutes,most preferably about an additional 45 minutes, during which time thebacterial cells are converted into spheroplasts and the intracellularproteins, including thermostable enzymes, begin to permeate thespheroplast membrane while cellular nucleic acids are retained withinthe spheroplast.

Microfiltration, Concentration and Diafiltration

Following permeabilization of the bacterial cells, thermostable enzymesare collected by subjecting the spheroplasts to microfiltration torelease the enzymes from the spheroplasts and remove particulate matter,concentration of the microfiltrate, and diafiltration. Unlike othermethods described in the background of the invention, the presentmethods obviate the need for precipitation of nucleic acids and the useof centrifugation techniques; this elimination of centrifugationfacilitates the rapid production of thermostable enzymes at any scale ina continuous or discontinuous fashion. The general methods ofmicrofiltration, concentration and diafiltration are generallywell-known to one of ordinary skill, and will result in the preparationof a nucleic acid-free enzyme ultrafiltrate suitable for purificationand characterization of the enzymes.

Microfiltration is preferably carried out by collecting the spheroplastsolution in permeabilization buffer (described above) and diafilteringthe solution against a filtration buffer through a semi-permeablemembrane, most preferably a hydrophilic dialysis, microfiltration orultrafiltration membrane. The filtration buffer preferably is adH₂O-based solution comprising: a) a buffer salt, preferablytrishydroxymethylaminomethane (TRIS® base) at a concentration of about25-500 mM, preferably about 50-250 mM, more preferably about 50-150 mM,and most preferably about 100 mM, at a pH of about 7.5-9.5, preferablyabout 8.0-9.3, more preferably about 8.0-9.0, and most preferably about8.9 (pH at 4° C.); and b) the protein-extracting salt which was added tothe permeabilization buffer, which is preferably ammonium sulfate orpotassium chloride, at a concentration of about 100-500 mM, preferablyabout 100-300 mM, and most preferably about 200 mM. The filtrationbuffer solution may also comprise other components, such as proteaseinhibitors (e.g., phenylmethylsulfonylfluoride, added at a finalconcentration of about 0.5 mM), reducing agents (e.g., β-mercaptoethanolor most preferably dithiothreitol at a final concentration of about 1mM), and chelating agents (e.g., disodium ethylenediaminetetraaceticacid (Na₂EDTA), most preferably at a concentration of about 1.5 mM);this buffer composition is referred to hereinafter as “filtrationbuffer.” It will be understood by one of ordinary skill in the art,however, that other suitable buffer compositions may be substituted withequivalent effect in the filtration process, provided that theabove-described pH levels and concentration of protein-extracting saltare used.

Preferable for use in microfiltration is a system allowing permeation ofintracellular enzymes through the membrane and into the filtrate,leaving spheroplasts (with the nucleic acids retained therein) andparticulate matter in the retentate. One suitable system providing suchconditions is, for example, a hollow fiber microfiltration system whichis commercially available (Microgon), although similar systems providingthe same results will be known to one of ordinary skill. Followingmicrofiltration in this manner, the filtrate contains the thermostableenzymes which are substantially free of nucleic acids such as DNA, asthe DNA is partitioned from the enzymes by being retained with theparticulate matter. This filtrate may then be concentrated, for exampleby membrane concentration through a semi-permeable membrane using acommercially available system (Amicon) or equivalent. The enzymes maythen be individually purified from the concentrate as described below;alternatively, the concentrate may be diafiltered as described aboveagainst a suitable buffer solution to place the enzymes into anappropriate chemical environment for purification, as described in moredetail in Example 2.

Purification and Characterization of Enzymes

Following concentration or diafiltration as described above,thermostable enzymes may be purified by a variety of proteinpurification techniques that are well-known to one of ordinary skill inthe art. Suitable techniques for purification include, but are notlimited to, ammonium sulfate or ethanol precipitation, acid extraction,preparative gel electrophoresis, immunoadsorption, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, immunoaffinitychromatography, size exclusion chromatography, liquid chromatography(LC), high performance LC (HPLC), fast performance LC (FPLC),hydroxylapatite chromatography and lectin chromatography. Mostpreferably, the enzymes are purified by a combination of liquidchromatographic techniques including ion exchange, affinity and sizeexclusion methods such as those described in Example 3, althoughalternative chromatographic solid supports, mobile phases and associatedmethods may be equivalently used and will be well-known to one ofordinary skill. Since all materials and equipment used in thechromatographic purification of the enzymes are sterilized or preparedso as to eliminate bacterial contamination, the enzyme preparations ofthermostable enzymes produced by this purification scheme will remainsubstantially free of nucleic acids such as DNA.

Assays for DNA Content

Purified thermostable enzymes made according to the present inventionmay be examined for nucleic acid content by a variety of methods whichare well-known to one of ordinary skill in the art. For example, asample of the final product can be assayed by ultravioletspectrophotometry, comparing absorption of light by the sample at awavelength of 260 nm (A₂₆₀, the absorption maximum for DNA) to that at280 nm (A₂₈₀, the absorption maximum for tryptophan, which is found inmost proteins); the lower the A₂₆₀/A₂₈₀ ratio, the lower the content ofDNA in the sample. Samples with minimal A₂₆₀/A₂₈₀ values may then bepooled to constitute a substantially nucleic acid-free preparation ofthermostable enzymes.

Alternatively, samples may be directly assayed for the presence of DNAby gel electrophoresis or dot blotting and staining with a DNA-bindingdye (e.g., ethidium bromide, acridine orange, Hoechst stain) orantibody, which are commercially available, for example, from Sigma (St.Louis, Mo.). In addition, the DNA content of samples of DNA polymerasesmay be examined by carrying out an amplification reaction in the absenceof exogenously added DNA template, either as a “no-template control” ina standard PCR assay (Rand, K. H., and Houck, H., Mol. Cell Probes4(6):445-450 (1990)), or by specifically designing an assay to measureDNA content by radiolabeled nucleotide incorporation into salmon testesor bovine thymus DNA, according to methods that are standard in the art.Use of such assays will allow one of ordinary skill, without undueexperimentation, to identify samples of thermostable enzymes obtained bythe purification schemes described above, which may then be pooled andused as preparations of substantially nucleic acid-free thermostableenzymes.

Formulation of Enzymes

Following their purification, the substantially DNA-free thermostableenzymes may be stored until use in a buffered solution at temperaturesof about −80° to 25° C., most preferably at −80° to 4° C., or inlyophilized form. Alternatively, the enzymes may be stabilized by dryingin the presence of a sugar such as trehalose (U.S. Pat. Nos. 5,098,893and 4,824,938) or acacia gum, pectin, carboxymethylcellulose,carboxymethylhydroxyethylcellulose, guar, carboxy guar,carboxymethylhydroxypropyl guar, laminaran, chitin, alginates orcarrageenan. In addition, the enzymes provided by the present inventionmay be directly formulated into compositions to be used in techniquesrequiring the use of thermostable enzymes, such as compositions fornucleic acid sequencing or amplification in the case of thermostable DNApolymerases such as Taq, Tne, or Tma DNA polymerases, or mutants,derivatives or fragments thereof. These formulations may be concentratedsolutions of the enzymes, or solutions of the enzymes at workingconcentrations which may comprise additional components and which may beprepared as described in co-pending U.S. patent application Ser. No.08/689,815, by Ayoub Rashtchian and Joseph Solus, entitled “StableCompositions for Nucleic Acid Sequencing and Amplification,” filed Aug.14, 1996, which is incorporated by reference herein in its entirety.

Kits

In other preferred embodiments, the substantially DNA-free thermostableenzymes provided by the present invention may be assembled into kits foruse in methods requiring thermostable enzymes, such as nucleic acidamplification or sequencing utilizing thermostable DNA polymerases suchas Taq, Tne or Tma DNA polymerases or mutants, fragments or derivativesthereof. A DNA amplification kit according to the present inventioncomprises a carrier means having in close confinement therein one ormore container means, such as vials, tubes, bottles and the like,wherein a first container means contains a substantially DNA-freethermostable enzyme, preferably a substantially DNA-free thermostableDNA polymerase or a thermostable restriction enzyme, and most preferablya substantially DNA-free preparation of Taq DNA polymerase, Tne DNApolymerase, Tma DNA polymerase, or a mutant, derivative or fragmentthereof. The amplification kit encompassed by this aspect of the presentinvention may further comprise additional reagents and compoundsnecessary for carrying out standard nucleic amplification protocols (SeeU.S. Pat. Nos. 4,683,195 and 4,683,202, which are directed to methods ofDNA amplification by PCR).

Similarly, a DNA sequencing kit according to the present inventioncomprises a carrier means having in close confinement therein one ormore container means, such as vials, tubes, bottles and the like,wherein a first container means contains a substantially DNA-freethermostable enzyme, preferably a substantially DNA-free thermostableDNA polymerase or a thermostable restriction enzyme, and most preferablya substantially DNA-free preparation of Taq DNA polymerase, Tne DNApolymerase, Tma DNA polymerase, or a mutant, derivative or fragmentthereof. The sequencing kit may further comprise additional reagents andcompounds necessary for carrying out standard nucleic sequencingprotocols, such as pyrophosphatase, agarose or polyacrylamide media forformulating sequencing gels, and other components necessary fordetection of sequenced nucleic acids (See U.S. Pat. Nos. 4,962,020 and5,498,523, which are directed to methods of DNA sequencing).

Use of the Substantially DNA-free Thermostable Enzymes

The substantially DNA-free thermostable enzymes and kits embodied in thepresent invention will have general utility in any application utilizingthermostable enzymes, including but not limited to nucleic acidamplification or sequencing methodologies. Amplification methods inwhich the present enzymes may be used include PCR (U.S. Pat. Nos.4,683,195 and 4,683,202), Strand Displacement Amplification (SDA; U.S.Pat. No. 5,455,166; EP 0 684 315), and Nucleic Acid Sequence-BasedAmplification (NASBA; U.S. Pat. No. 5,409,818; EP 0 329 822). Nucleicacid sequencing techniques which may employ the present enzymes includedideoxy sequencing methods such as those disclosed in U.S. Pat. Nos.4,962,022 and 5,498,523, as well as more complex PCR-based nucleic acidfingerprinting techniques such as Random Amplified Polymorphic DNA(RAPD) analysis (Williams, J. G. K., et al., Nucl. Acids Res.18(22):6531-6535, 1990), Arbitrarily Primed PCR (AP-PCR; Welsh, J., andMcClelland, M., Nucl. Acids Res. 18(24):7213-7218, 1990), DNAAmplification Fingerprinting (DAF; Caetano-Anollés et al.,Bio/Technology 9:553-557, 1991), microsatellite PCR or DirectedAmplification of Minisatellite-region DNA (DAMD; Heath, D. D., et al.,Nucl. Acids Res. 21(24): 5782-5785, 1993), and Amplification FragmentLength Polymorphism (AFLP) analysis EP 0 534 858; Vos, P., et al., Nucl.Acids Res. 23(21):4407-4414, 1995; Lin, J. J., and Kuo, J., FOCUS17(2):66-70, 1995). In particular, the enzymes and kits of the presentinvention will be useful in the fields of medical therapeutics anddiagnostics, forensics, and agricultural and other biological sciences,in any procedure utilizing thermostable enzymes such as thermostable DNApolymerases or thermostable restriction endonucleases.

It will be readily apparent to those skilled in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Example 1 Permeabilization of Bacterial Cells

In the initial steps of the purification process, bacterial cells(Thermus aquaticus, strain YT-1), which were obtained directly fromactively growing cultures, or which were stored at −20° C. and partiallythawed, were suspended at 500 g of cells/L into cold (4° C.,) ofpermeabilization buffer (100 mM TRIS, 1.8% TRITON X-100, 600 mMguanidine HCl, 1.5 mM EDTA, 1 mM dithiothreitol (DTT), pH 8.5). Duringsuspension of the cells in the buffer, phenylmethylsulfonylfluoride(PMSF) was added to a final concentration of 0.5 mM. Cells were stirredfor about 45 minutes at 4° C. to ensure complete suspension, and thenammonium sulfate was added to a final concentration of 200 mM and thecell suspension was stirred for an additional 45 minutes. During thistime, cells were permeabilized via the action of the guanidine HCl andTRITON X-100, and intracellular protein release into the buffer wasenhanced by the action of the ammonium sulfate.

Example 2 Microfiltration, Concentration and Diafilitration of Extracts

Following permeabilization, one volume of filtration buffer (100 mMTRIS, 200 mM ammonium sulfate, 1 mM EDTA, 1 mM DTT, pH 8.9) was added tothe cell suspension, and the suspension mixed completely.Microfiltration of the suspension was then carried out through a 0.2 μmMicrogon mixed ester cellulose hollow fiber system, using arecirculation rate of 90 L/min. The suspension was diafiltered with sixvolumes of cold filtration buffer, collecting the permeate in a suitablesized chilled (4° C.) container. Under these conditions, thermostableenzymes passed through the membrane with the permeate, leaving thebacterial cells with most of the DNA in the retentate.

As the ultrafiltration proceeded, concentration of the permeate wasbegun once a sufficient volume had been collected to prime the secondultrafiltration system. Permeate was concentrated using an Amicon DC-90system, through Microgon 10,000 MWCO membrane (although alternativemembrane systems of 10,000 MWCO, such as a Filtron system, a Milliporeplate and frame system, or membrane from AG Technologies, may be usedequivalently) and an in-line chiller to minimize heat build-up from thepumping system. Permeate was concentrated to approximately the originalvolume of the extract (see Example 1), and was then diafiltered againstabout seven volumes of diafiltration buffer (25 mM Bicine, 10 mM KCl, 1mM EDTA, 1 mM DTT, pH 8.9), until the conductivity was ≦3 mS.Ultrafiltrate was then immediately used for purification of enzyme(Example 3), or was stored at −80° to 4° C. until use. For storage,ultrafiltrate is most preferably lyophilized and stored at −20° C.,although ultrafiltrate solution may alternatively be formulated into 50%(w/v) glycerol and stored at −20° C. or pre-loaded onto Fractogel SO₃matrix (see Example 3A below) and stored at 4° C. until use.

Example 3 Purification and Characterization of DNA-free Taq DNAPolymerase

Purification of enzyme from ultrafiltrate was accomplished by a seriesof chromatographic steps, using a procedure modified slightly from thatdescribed for purification of T5 DNA polymerase from E. coli (Hughes, A.J., Jr., et al., J. Cell. Biochem. Suppl. 0 16(Part B):84 (1992)).

A. Fractogel SO₃

In the first chromatographic step, ultrafiltrate was passed over aFractogel EMD SO₃ ⁻-650(M) column (EM Separation Technologies, whichremoves most of the Taq I restriction enzyme and crude proteins from theultrafiltrate. Ultrafiltrate was passed through a polypropylene depthfilter to remove any residual precipitation, and loaded onto theFractogel column at a flow rate of about 30 cm/hr. The column was thenwashed with 10 volumes of diafiltration buffer, and a 20 volume gradientfrom diafiltration buffer to SO₃ elution buffer (25 mM bicine, 500 mMKCl, 1 mM EDTA, 1 mM DTT, pH 8.9) run at a flow rate of about 30 cm/hr.Fractions were then collected for assay in diafiltration buffer for TaqDNA polymerase activity, starting with those fractions corresponding tothe major UV peak. Assays were performed as described below undersection F entitled “Cross-column Assays.” Fractions demonstrating atleast ½ of peak polymerase activity were pooled and subjected to furtherpurification.

B. Single-stranded DNA Agarose Column

Pooled eluate from the Fractogel column was concentrated using aPellicon 5 ft² apparatus, 10,000 MWCO with a T-screen Filtron cassetteand a flow rate of 3 L/min at 10 psi until the volume was about ½ thestarting volume. The sample was then diafiltered with low salt SS DNAagarose buffer (25 mM K₂PO₄, 10 mM KCl, 1 mM EDTA, 10% (vol/vol)glycerol, 1 mM DTT, pH 6.5) until the conductivity of the retentatereached <3.5 mS at 4° C. Sample was applied to a SS DNA agarose column(LTI; Gaithersburg, Md.) at a flow-rate of 10 cm/hour, and the columnwas then washed with 10 volumes of low salt SS DNA agarose buffer at aflow-rate of 20 cm/hour. Enzyme was then eluted with a 20 column volumegradient from low salt SS DNA agarose buffer to high salt SS DNA agarosebuffer (750 mM KCl) at a flow rate of 10 cm/hr. Fractions were collectedas described above for Taq DNA polymerase activity assay, and fractionsshowing at least ½ of peak activity were pooled (the enzyme usuallyelutes between 30 and 40% of the gradient) and subjected to furtherpurification.

C. Ceramic Hydroxyapatite, Type A

Sample from the SS DNA agarose column was applied to a ceramichydroxyapatite column (AIC, Inc.), which removes residual endonucleasesand other non-specific proteins, at a flow rate of 10 cm/hr. The columnwas then washed with 1-2 column volumes of low salt ceramic HTP buffer(20 mM K₂PO₄, 100 mM KCl, 10% glycerol, 1 mM DTT, pH 6.5), and theenzyme eluted with a 10 column volume linear gradient of low saltceramic HTP buffer to high salt ceramic HTP buffer (500 mM K₂PO₄) at 10cm/hr, and fractions collected, assayed and pooled as described above(the enzyme usually elutes as a major UV peak 35 to 45% through thegradient).

D. AF-Heparin-650M

Eluate from the ceramic hydroxyapatite column was then applied to anAF-Heparain-650M affinity column (TosoHaas), which removes residualproteins and results in single-band purity Taq polymerase.

Pooled eluate from the ceramic hydroxyapatite column was diafilteredagainst five volumes of low salt AF-heparin buffer (25 mM K2PO4, 10 mMKCl, 1 mM EDTA, 1 mM DTT, pH 6.5) using a Filtron system with aT-screen, 10,000 MWCO omega membrane cassette until the conductivitylevel reached. ≦5 mS at 4° C. If a precipitate appeared, sample wasfiltered thru a 0.4-5 μm low protein-binding membrane filter prior toloading onto the column. Sample was loaded onto the AF-Heparin column ata flow rate of 25 cm/hr, and the column then washed with 10 columnvolumes of low salt AF-heparin buffer at 25 cm/hr. Enzyme was theneluted with a 20 column volume gradient of low salt AF-heparin buffer tohigh salt AF-heparin buffer (750 mM KCl) at 25 cm/hr. Fractions werecollected, assayed and pooled as described above (the enzyme typicallyeluted as a major UV peak ˜40 to 45% of the gradient).

E. Dialysis

AF-Heparin pool was dialyzed against 12 volumes of dialysis buffer (20mM TRIS, 0.1 mM EDTA, 50% (vol/vol) glycerol, 1 mM DTT, pH 8.0) for 5hours, then dialyzed overnight against 12 volumes of fresh dialysisbuffer. Following dialysis, an equal volume of dialysis buffer was addedto the dialyzed pool, and the pool mixed by magnetic stirring for about1 hour at 4° C. Purified enzyme bulk was then stored at −20° C. untiluse.

F. Across-column Assays:

To determine polymerase activity in fractions at each step of thepurification process, an assay modified from that described previously(Lehman, I. R., et al., J. Biol. Chem. 233(1):163-170 (1958)) was used.Briefly, tubes containing a reaction mixture (30 μl each) were prepared,with each tube containing 5 μl of column fraction sample in 25 μl ofassay buffer (25 mM TAPS (pH 9.3), 2 mM MgCl₂, 50 mM KCl, 1 mM DTT, 0.2mM each of dATP, dTTP, dGTP and dCTP, 0.4 mCi/ml of [³H]dTTP (40Ci/mmol), and 500 μg/ml of activated salmon testes DNA). Samples werevortexed gently and incubated at 72° C. for 10 minutes. Afterincubation, tubes were placed on ice for five minutes, microcentrifugedfor five seconds, and then a 20 μl sample from each tube was spottedonto a GF/C filter and washed with cold 10% (w/v) trichloroacetic acid(TCA)/1% (w/v) sodium pyrophosphate for five minutes, followed by threewashes with 5% TCA for three minutes each, and two washes in absoluteethanol for three minutes each. The filters were then dried under a heatlamp for fifteen minutes and then placed into Econofluor and quantifiedfor ³H in a Beckman scintillation counter.

G. DNA Contamination Assays:

To determine the extent of DNA contamination of various preparations ofTaq DNA polymerase, samples of Taq DNA polymerase obtained fromcommercial sources were compared to a preparation made according to themethods of the present invention in a polymerase assay similar to thatoutlined above, except that no salmon testes DNA template was includedin the reaction mixture. Briefly, reaction mixtures (500 μl) containing25 mM TAPS (pH 9.3), 10 mM MgCl₂, 50 mM KCl, 1 mM DTT, 100 μM each ofdATP, dTTP, dGTP and dCTP, and 600 cpm of [³H]dTTP/pmol of totalnucleotide, were prepared and preincubated at 72° C. for five minutes.100 units of purified Taq DNA polymerase was added to initate thereaction and at the times indicated in FIGS. 1 and 2, a 30 μl sample wasremoved and added to a vial containing 5 μl of 500 mM EDTA on ice. Onceall time points had been collected, a 20 μl aliquot of the quenchedreaction sample was applied to a GF/C filter, which were washed, driedand counted as described above. Results were expressed as ³Hincorporation (cpm) at each time point.

As shown in FIG. 1, a sample of purified Taq DNA polymerase (i.e.,dialysed eluate from the AF-Heparin column above) prepared according tothe methods of the present invention (“New Taq”) demonstrated nodetectable incorporation of [³H]TTP over a period of incubation of 120minutes. A commercial preparation of Taq polymerase made according totraditional methods (“Old Taq”), however, showed significant nucleotideincorporation beginning at 80 minutes of incubation, and increasingthereafter. These results indicate that preparations of Taq DNApolymerase provided by the present invention do not incorporatenucleotides in the absence of exogenously added template DNA,demonstrating that the present preparations of Taq DNA polymerase aresubstantially free from contamination with nucleic acids, while thoseobtained by traditional manufacturing methods contain significantamounts of contaminating DNA.

To confirm these results, other commercially available preparations ofTaq DNA polymerase were compared to the preparations provided by thepresent invention for their DNA content. As shown in FIG. 2, commercialpreparations of Taq DNA polymerase obtained from two other vendors(“Promega,” “BM”) contained substantial amounts of contaminating DNA, asindicated by the significant incorporation of [³H]TTP in samplescontaining these preparations at reaction times greater than 80 minutes.As seen above, however, the present preparations of Taq DNA polymerase(“New Taq”) demonstrated no detectable incorporation of [³H]TTP over anincubation period of 120 minutes. Together, these results indicate thatpreparations of Taq DNA polymerase provided by the present invention aresubstantially free of nucleic acids, while several commonly usedcommercial preparations of Taq DNA polymerase contain substantialamounts of contaminating DNA.

Having now fully described the present invention it will be understoodby those of ordinary skill in the art that the same can be performedwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof.

All publications, patents and patent applications cited herein areindicative of the level of skill of those skilled in the art to whichthis invention pertains, and are herein incorporated by reference intheir entirety.

1. A method for permeabilizing a bacterium, said method comprisingsuspending one or more bacterial cells that produce at least onethermostable enzyme in a permeabilization buffer, said buffer comprisingat least one chaeotropic agent and at least one non-ionic detergent,thereby producing permeabilized bacterial cells that retain DNA andallow free permeation of intracellular proteins across their membrane.2. The method of claim 1, wherein said bacterial cells produce athermostable DNA polymerase.
 3. The method of claim 2, wherein saidthermostable DNA polymerase is selected from the group consisting of TaqDNA polymerase, Tne DNA polymerase, Tma DNA polymerase, and mutants,derivatives or fragments thereof having DNA polymerase activity.
 4. Themethod of claim 2, wherein said thermostable DNA polymerase is Taq DNApolymerase.
 5. The method of claim 1, wherein said bacterial cellsproduce a thermostable restriction endonuclease.
 6. The method of claim1, wherein said permeabilization buffer further comprises one or moreingredients selected from the group consisting oftrishydroxymethylaminomethane (TRIS base), one or more proteaseinhibitors, one or more reducing agents and one or more chelatingagents.
 7. The method of claim 1, wherein said permeabilization bufferfurther comprises phenylmethylsulfonylfluoride.
 8. The method of claim1, wherein said permeabilization buffer further comprisesβ-mercaptoethanol or dithiothreitol.
 9. The method of claim 1, whereinsaid permeabilization buffer further comprises disodiumethylenediaminetetraacetic acid (Na₂EDTA).
 10. The method of claim 1,said method further comprising adding one or more protein-extractingsalts to said one or more bacterial cells after being suspended in saidpermeabilization buffer.
 11. The method of claim 10, wherein saidprotein-extracting salt is selected from the group consisting of sodiumchloride, potassium acetate, sodium acetate, ammonium acetate, ammoniumchloride, ammonium sulfate, and potassium chloride.
 12. The method ofclaim 1, wherein said bacterial cells are suspended in saidpermeabilization buffer at a concentration of about 100 g to about 1000g (wet weight) of cells per liter of buffer.
 13. The method of claim 12,wherein said bacterial cells are suspended in said permeabilizationbuffer at a concentration of about 250 g to about 1000 g (wet weight) ofcells per liter of buffer.
 14. The method of claim 13, wherein saidbacterial cells are suspended in said permeabilization buffer at aconcentration of about 500 g (wet weight) of cells per liter of buffer.15. The method of claim 1, wherein said chaeotropic agent is a salt ofguanidine or urea.
 16. The method of claim 1, wherein said chaeotropicagent is guanidine hydrochloride.
 17. The method of claim 1, wherein theconcentration of said chaeotropic agent in said buffer is about 300 mMto about 1000 mM.
 18. The method of claim 17, wherein the concentrationof said chaeotropic agent in said buffer is about 500 mM to about 750mM.
 19. The method of claim 18, wherein the concentration of saidchaeotropic agent in said buffer is about 600 mM.
 20. The method ofclaim 1, wherein said non-ionic detergent isoctylphenoxypolyethoxyethanol nonionic surfactant (TRITON X-100), Brij35, Tween 20 or Nonidet P-40 (NP-40).
 21. The method of claim 1, whereinsaid non-ionic detergent is selected from the group consisting of anN-alkylglucoside, an N-akylmaltoside, a glucamide, digitonin,deoxycholate,3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) andcetyltrimethylammoniumbromide (CTAB).
 22. The of method claim 1, whereinthe concentration of said non-ionic detergent in said buffer is about 1%to about 5% (vol/vol).
 23. The method of claim 22, wherein theconcentration of said non-ionic detergent in said buffer is about 1% toabout 2.5% (vol/vol).
 24. The method of claim 23, wherein theconcentration of said non-ionic detergent in said buffer is about 1.8%.